PROJECT SUMMARY Adult stem cells hold significant therapeutic potential to treat many diseases and injuries. For example, neural progenitor cells (NPCs) are currently being investigated in over 20 clinical trials for use in a variety of indications. Despite their significant clinical relevance, we currently lack the biological mechanistic understanding to efficiently expand NPCs in vitro, even as neurospheres, while maintaining their undifferentiated, regenerative stem phenotype. Recently, 3D matrices have emerged as a tool for stem cell expansion; unfortunately, once encapsulated, NPCs commonly lose their stemness and ability to proliferate. Loss of NPC stemness is also observed in vivo throughout the aging process and in pathological disease states causing diminished ability for NPC self-renewal and biased differentiation. These phenotypic abnormalities are due in part to complex environmental changes in the stem cell niche including altered extracellular matrix biochemical and biomechanical properties. Therefore, we propose the use of a 3D in vitro hydrogel culture platform with controlled matrix biochemistry and biomechanics that will enable the exploration of previously untestable hypotheses on the mechanisms by which the surrounding cell microenvironment influences NPC maintenance, expansion, and differentiation. We will use a family of protein-engineered hydrogels to understand the impact of the matrix microenvironment on human iPSC-derived NPC (hNPC) phenotype. Specifically, we will study the role of matrix biochemical and biomechanical properties on activation of the N-cadherin signaling pathway and downstream hNPC phenotype. In Aim 1, we tune the biochemical cues presented within elastin-like protein (ELP) hydrogels to display a N-cadherin-mimetic peptide. We hypothesize that cell engagement with the artificial N-cadherin will result in downstream ?-catenin signaling, stemness maintenance, and enhanced symmetric proliferation compared to neurosphere controls. In Aim 2, we tune the biomechanical cues presented by the recombinant ELP hydrogels to enable dynamic matrix remodeling through viscoelastic stress relaxation. We hypothesize that dynamic matrix remodeling will result in increased cell-cell contacts, induction of cellular-based N-cadherin signaling, stemness maintenance, and enhanced symmetric proliferation compared to neurosphere controls. In Aim 3, we evaluate the hypothesis that control of specific matrix material properties to tune N-cadherin presentation and ELP hydrogel mechanics alters outside-in signal transduction that biases hNPC differentiation. The biological mechanisms underlying this process will be explored via changes in nuclear architecture (lamin expression and nuclear morphology) and epigenetics (histone modification and chromosomal organization). Further mechanistic insight will be explored using inhibitors and agonists of key mechanotransduction signaling pathways. Our engineered, modular hydrogels allow us to explore the mechanisms by which specific matrix cues regulate hNPC stem maintenance and differentiation. Given the immense regenerative potential of these cells, our findings will inform the design of a robust in vitro platform for the clinical expansion of hNPCs.